Modification of Flavonoid Biosynthesis in Plants

ABSTRACT

The present invention relates to nucleic acids encoding flavonoid biosynthetic enzymes, flavonoid-regulating transcription factors and a flavonoid-specific membrane transporter in plants, and the use thereof for the modification of flavonoid biosynthesis in plants. The present invention also relates to constructs and vectors including such nucleic acids, and related polypeptides. More particularly, the protein involved in flavonoid biosynthesis is selected from the group consisting of TRANSPARENT TESTA 12 (TT12), TRANSPARENT TESTA GLABRA 1 (TTG1), TRANSPARENT TESTA 2 (TT2), TRANSPARENT TESTA 8 (TT8), leucoanthocyanidin dioxygenase (LDOX), cinnamate-4-hydroxylase (C4H), 4-coumaroyl:CoA-ligase (4CL); and functionally active fragments and variants thereof.

The present invention relates generally to nucleic acid fragments andtheir encoded amino acid sequences for flavonoid biosynthetic enzymes inplants, and the use thereof for the modification of flavonoidbiosynthesis in plants.

Flavonoids constitute a relatively diverse family of aromatic moleculesthat are derived from phenylalanine and malonyl-coenzyme A (CoA, via thefatty acid pathway). These compounds include six major subgroups thatare found in most higher plants: the chalcones, flavones, flavonols,flavandiols, anthocyanins and condensed tannins (or proanthocyanidins).A seventh group, the aurones, is widespread, but not ubiquitous.

Some plant species also synthesize specialised forms of flavonoids, suchas the isoflavonoids that are found in legumes and a small number ofnon-legume plants. Similarly, sorghum and maize are among the fewspecies known to synthesize 3-deoxyanthocyanins (or phlobaphenes in thepolymerised form). The stilbenes, which are closely related toflavonoids, are synthesised by another group of unrelated species thatincludes grape, peanut and pine.

Besides providing pigmentation to flowers, fruits, seeds, and leaves,flavonoids also have key roles in signalling between plants andmicrobes, in male fertility of some species, in defense as antimicrobialagents and feeding deterrants, and in UV protection.

Flavonoids also have significant activities when ingested by animals,and there is great interest in their potential health benefits,particularly for compounds such as isoflavonoids, which have been linkedto anticancer benefits, and stilbenes that are believed to contribute toreduced heart disease.

The major branch pathways of flavonoid biosynthesis start with generalphenylpropanoid metabolism and lead to the nine major subgroups: thecolorless chalcones, aurones, isoflavonoids, flavones, flavonols,flavandiols, anthocyanins, condensed tannins, and phlobaphene pigments.The enzyme phenylalanine ammonialyase (PAL) of the generalphenylpropanoid pathway will lead to the production of cinnamic acid.Cinnamate-4-hydroxylase (C4H) will produce p-coumaric acid which will beconverted through the action of 4-coumaroyl:CoA-ligase (4CL) to theproduction of 4-coumaroyl-CoA and malonyl-CoA.

In the phenylpropanoid pathway, chalcone synthase (CHS) uses malonyl CoAand 4-coumaryl CoA as substrates. Chalcone reductase (CHR) balances theproduction of 5-hydroxy- or 5-deoxyflavonoids. The next enzyme, chalconeisomerase (CHI) catalyses ring closure to form a flavanone, but thereaction can also occur spontaneously. Further enzymes in the pathwayare: flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR),flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′, 5′ hydroxylase(F3′5′H).

In the branch of the phenylpropanoid pathway that is specific tocondensed tannin and anthocyanin production, leucoanthocyanidins can bereduced to catechins by leucoanthocyanidin reductase (LAR) or toanthocyanidins by leucoanthocyanidin dioxygenase (LDOX). Anthocyanidinscan be converted to anthocyanins by the addition of sugar groups, or toepicatechins by anthocyanidin reductase (ANR), encoded by the BANYULSgene. Catechins and epicatechins are the subunits of condensed tannins(CTs), which in Arabidopsis are thought to be transported into thevacuole by a multidrug secondary transporter-like protein, TRANSPARENTTESTA 12 (TT12), and polymerised by an unknown mechanism.

Enzymes in the flavonoid pathway have been found to be controlled by arange of transcription factors in Arabidopsis, maize and petunia. InArabidopsis, condensed tannin biosynthesis requires the function ofTRANSPARENT TESTA 2 (TT2), a myb family factor, TRANSPARENT TESTA 8(TT8), a myc family factor and TRANSPARENT TESTA GLABRA 1 (TTG1), a WD40family factor, among other transcription factors. These three proteinsare thought to form a transcription complex that coordinately activatesmultiple flavonoid pathway enzymes in order to promote condensed tanninproduction in Arabidopsis seeds. Other myc and myb family transcriptionfactors regulate distinct parts of the flavonoid pathway in maize,petunia and other plant species.

While nucleic acid sequences encoding some flavonoid biosyntheticenzymes have been isolated for certain species of plants, for examplecertain C4H, 4CL, LDOX, TT12-like transporters and TT8-like, TT4-likeand TTG1-like transcription factors, there remains a need for materialsuseful in modifying flavonoid biosynthesis; in modifying proteinbinding, metal chelation, anti-oxidation, and UV-light absorption; inmodifying plant pigment production; in modifying plant defense to bioticstresses such as viruses, microorganisms, insects, fungal pathogens; inmodifying forage quality, for example by disrupting protein foam andconferring protection from rumen pasture bloat, particularly in foragelegumes and grasses, including alfalfa, medics, clovers, ryegrasses andfescues. There is also a need for methods of using such materials.

It is an object of the present invention to overcome, or at leastalleviate, one or more of the difficulties or deficiencies associatedwith the prior art or to assist in meeting the needs stated above.

In one aspect, the present invention provides a substantially purifiedor isolated nucleic acid or nucleic acid fragment encoding a flavonoidbiosynthesis-regulating transcription factor selected from the groupconsisting of TRANSPARENT TESTA GLABRA 1 (TTG1), TRANSPARENT TESTA 2(TT2), and TRANSPARENT TESTA 8 (TT8); a flavonoid biosynthetic enzymeselected from the group consisting of leucoanthocyanidin dioxygenase(LDOX), cinnamate-4-hydroxylase (C4H) and 4-coumaroyl:CoA-ligase (4CL);and a flavonoid transporter TRANSPARENT TESTA 12 (TT12); from a clover(Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca)species; or a functionally active fragment or variant thereof. Thepresent invention further provides substantially purified or isolatednucleic acids or nucleic acid fragments complementary and antisense tothe nucleic acids or nucleic acid fragments of the present invention.

The present invention also provides substantially purified or isolatednucleic acids or nucleic acid fragments encoding amino acid sequencesfor a class of proteins which are related to C4H, 4CL, LDOX, TT12, TT2,TT8 and TTG1, or functionally active fragments or variants thereof. Suchproteins are referred to herein as C4H-like, 4CL-like, LDOX-like,TT12-like, TT2-like, TT8-like and TTG1-like, respectively. Proteins arerelated in that either one of both of the following criteria apply: (i)the genes which encode these proteins are expressed in a similar mannerto C4H, 4CL, LDOX, TT12, TT2, TT8 or TTG1, and (ii) the polypeptideshave similar functional activity to C4H, 4CL, LDOX, TT12, TT2, TT8 andTTG1. In a preferred embodiment, the related proteins are at least 70%,preferably at least 80%, more preferably at least 90% homologous to C4H,4CL, LDOX, TT12, TT2, TT8 or TTG1. Also provided are substantiallyisolated nucleic acids or nucleic acid fragments complementary andantisense to C4H-like, 4CL-like, LDOX-like, TT12-like, TT2-like,TT8-like and TTG1-like-encoding nucleic acid fragments.

The individual or simultaneous enhancement or otherwise manipulation ofthe expression of C4H, 4CL, LDOX, TT12, TT2, TT8, TTG1 or -likepolypeptides in plants may enhance or otherwise alter flavonoidbiosynthesis; may enhance or otherwise alter the plant capacity forprotein binding, metal chelation, anti-oxidation, and UV-lightabsorption; may enhance or reduce or otherwise alter plant pigmentproduction.

The individual or simultaneous enhancement or otherwise manipulation ofthe expression of C4H, 4CL, LDOX, TT12, TT2, TT8, TTG1 or -likepolypeptides in plants has significant consequences for a range ofapplications in, for example, plant production and plant protection. Forexample, it has applications in increasing plant tolerance and plantdefense to biotic stresses such as viruses, microorganisms, insects andfungal pathogens; in improving plant forage quality, for example bydisrupting protein foam and in conferring protection from rumen pasturebloat; in reducing digestion rates in the rumen and reducing parasiticload; in the production of plant compounds leading to health benefits,such as isoflavonoids, which have been linked to anticancer benefits,and stilbenes that are believed to contribute to reduced heart disease.

White clover expresses multiple isoforms of 4CL and C4H. Co-ordinateexpression of genes encoding isoforms of 4CL, PAL and C4H that areinvolved in the production of specific flavonoids, such as CTs, mayallow the production of various flavonoids to be regulated independentlyby cell-specific factors and the circadian clock. Hence, theidentification of CT-specific isoforms of enzymes located early in thephenylpropanoid pathway is an important step towards modification ofthis pathway in forage legumes.

Methods for the manipulation of C4H, 4CL, LDOX, TT12, TT2, TT8, TTG1 orlike gene activities in plants, including legumes such as clovers(Trifolium species), lucerne (Medicago sativa) and grass species such asryegrasses (Lolium species) and fescues (Festuca species) may facilitatethe production of, for example, forage legumes and forage grasses andother crops with enhanced tolerance to biotic stresses such as viruses,microorganisms, insects and fungal pathogens; altered pigmentation inflowers; forage legumes with enhanced herbage quality and bloat-safety;crops with enhanced isoflavonoid content leading to health benefits.

The use of transcription factors to modify multiple product-specificenzymes in the flavonoid pathway may be a useful alternative strategy tocloning genes encoding many enzymes and modifying their expression intransgenic plants.

The clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue(Festuca) species may be of any suitable type, including white clover(Trifolium repens), red clover (Trifolium pratense), subterranean clover(Trifolium subterraneum), alfalfa (Medicago sativa), Italian or annualryegrass (Lolium multiflorum), perennial ryegrass (Lolium perenne), tallfescue (Festuca arundinacea), meadow fescue (Festuca pratensis) and redfescue (Festuca rubra). Preferably the species is a clover or aryegrass, more preferably white clover (T. repens) or perennial ryegrass(L. perenne). White clover (Trifolium repens L.) and perennial ryegrass(Lolium perenne L.) are key pasture legumes and grasses, respectively,in temperate climates throughout the world. Perennial ryegrass is alsoan important turf grass.

Nucleic acids according to the invention may be full-length genes orpart thereof, and are also referred to as “nucleic acid fragments” and“nucleotide sequences” in this specification. For convenience, theexpression “nucleic acid or nucleic acid fragment” is used to cover allof these.

The nucleic acid or nucleic acid fragment may be of any suitable typeand includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA)that is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases, and combinations thereof.

The term “isolated” means that the material is removed from its originalenvironment (eg. the natural environment if it is naturally occurring).For example, a naturally occurring nucleic acid present in a livingplant is not isolated, but the same nucleic acid separated from some orall of the coexisting materials in the natural system, is isolated. Suchnucleic acids could be part of a vector and/or such nucleic acids couldbe part of a composition, and still be isolated in that such a vector orcomposition is not part of its natural environment.

Such nucleic acids or nucleic acid fragments could be assembled to forma consensus contig. As used herein, the term “consensus contig” refersto a nucleotide sequence that is assembled from two or more constituentnucleotide sequences that share common or overlapping regions ofsequence homology. For example, the nucleotide sequence of two or morenucleic acids or nucleic acid fragments can be compared and aligned inorder to identify common or overlapping sequences. Where common oroverlapping sequences exist between two or more nucleic acids or nucleicacid fragments, the sequences (and thus their corresponding nucleicacids or nucleic acid fragments) can be assembled into a singlecontiguous nucleotide sequence.

In a preferred embodiment of this aspect of the invention, thesubstantially purified or isolated nucleic acid or nucleic acid fragmentencoding a TT12 or TT12-like protein or complementary or antisense to asequence encoding a TT12 or TT12-like protein includes a nucleotidesequence selected from the group consisting of (a) the sequences shownin FIGS. 1 and 33 hereto; (b) the complement of the sequences recited in(a); (c) sequences antisense to the sequences recited in (a) and (b);and (d) functionally active fragments and variants of the sequencesrecited in (a), (b) and (c).

In a further preferred embodiment of this aspect of the invention, thesubstantially purified or isolated nucleic acid or nucleic acid fragmentencoding a TTG1 or TTG1-like protein or complementary or antisense to asequence encoding a TTG1 or TTG1-like protein includes a nucleotidesequence selected from the group consisting of (a) the sequences shownin FIGS. 4 and 37 hereto; (b) the complement of the sequences recited in(a); (c) the sequence antisense to the sequences recited in (a) and (b);and (d) functionally active fragments and variants of the sequencesrecited in (a), (b) and (c).

In a further preferred embodiment of this aspect of the invention, thesubstantially purified or isolated nucleic acid or nucleic acid fragmentencoding an TT2 or TT2-like protein or complementary or antisense to asequence encoding a TT2 or TT2-like protein includes a nucleotidesequence selected from the group consisting of (a) sequences shown inFIGS. 6, 9, 41 and 44 hereto; (b) complements of the sequences recitedin (a); (c) sequences antisense to the sequences recited in (a) and (b);and (d) functionally active fragments and variants of the sequencesrecited in (a), (b) and (c).

In a further preferred embodiment of this aspect of the invention, thesubstantially purified or isolated nucleic acid or nucleic acid fragmentencoding a TT8 or TT8-like protein or complementary or antisense to asequence encoding a TT8 or TT8-like protein includes a nucleotidesequence selected from the group consisting of (a) the sequences shownin FIGS. 11 and 48 hereto; (b) the complement of the sequences recitedin (a); (c) the sequences antisense to the sequences recited in (a) and(b); and (d) functionally active fragments and variants of the sequencesrecited in (a), (b) and (c).

In a further preferred embodiment of this aspect of the invention, thesubstantially purified or isolated nucleic acid or nucleic acid fragmentencoding a LDOX or LDOX-like protein or complementary or antisense to asequence encoding a LDOX or LDOX-like protein includes a nucleotidesequence selected from the group consisting of (a) the sequences shownin FIGS. 13 and 52 hereto; (b) the complement of the sequences recitedin (a); (c) sequences antisense to the sequences recited in (a) and (b);and (d) functionally active fragments and variants of the sequencesrecited in (a), (b) and (c).

In a still further preferred embodiment of this aspect of the invention,the substantially purified or isolated nucleic acid or nucleic acidfragment encoding a 4CL or 4CL-like protein or complementary orantisense to a sequence encoding a 4CL or 4CL-like protein includes anucleotide sequence selected from the group consisting of (a) sequencesshown in FIGS. 16, 19, 21, 23, 56, 59, 62 and 65 hereto; (b) complementsof the sequences recited in (a); (c) sequences antisense to thesequences recited in (a) and (b); and (d) functionally active fragmentsand variants of the sequences recited in (a), (b) and (c).

In a further preferred embodiment of this aspect of the invention, thesubstantially purified or isolated nucleic acid or nucleic acid fragmentencoding a C4H or C4H-like protein or complementary or antisense to asequence encoding a C4H or C4H-like protein includes a nucleotidesequence selected from the group consisting of (a) sequences shown inFIGS. 25, 28, 30, 70, 74 and 77 hereto; (b) complements of the sequencesrecited in (a); (c) sequences antisense to the sequences recited in (a)and (b); and (d) functionally active fragments and variants of thesequences recited in (a), (b) and (c).

By “functionally active” in relation to nucleic acids it is meant thatthe fragment or variant (such as an analogue, derivative or mutant)encodes a polypeptide, which is capable of modifying flavonoidbiosynthesis; in a plant. Such variants include naturally occurringallelic variants and non-naturally occurring variants. Additions,deletions, substitutions and derivatizations of one or more of thenucleotides are contemplated so long as the modifications do not resultin loss of functional activity of the fragment or variant. Preferablythe functionally active fragment or variant has at least approximately75% identity to the relevant part of the above mentioned nucleotidesequence, more preferably at least approximately 80% identity, morepreferably at least approximately 90% identity, most preferably at leastapproximately 95% identity. Such functionally active variants andfragments include, for example, those having nucleic acid changes whichresult in conservative amino acid substitutions of one or more residuesin the corresponding amino acid sequence. Preferably the fragment has asize of at least 30 nucleotides, more preferably at least 45nucleotides, most preferably at least 60 nucleotides.

It will also be understood that the term “comprises” (or its grammaticalvariants) as used in this specification is equivalent to the term“includes” and should not be taken as excluding the presence of otherelements or features.

Nucleic acids or nucleic acid fragments encoding at least a portion ofseveral C4Hs, 4CLs, LDOXs, and candidate TT12, TT2, TT8 and TTG1orthologs have been isolated and identified. The nucleic acids ornucleic acid fragments of the present invention may be used to isolatecDNAs and genes encoding homologous proteins from the same or otherplant species. Isolation of homologous genes can be isolated usingsequence-dependent protocols, such as methods of nucleic acidhybridisation, and methods of DNA and RNA amplification as exemplifiedby various uses of nucleic acid amplification technologies (e.g.polymerase chain reaction, ligase chain reaction).

For example, genes encoding other C4H or C4H-like, 4CL or 4CL-like, LDOXor LDOX-like, TT12-like, TT2-like, TT8-like, TTG1-like proteins, eitheras cDNAs or genomic DNAs, may be isolated directly by using all or aportion of the nucleic acids or nucleic acid fragments of the presentinvention as hybridisation probes to screen libraries from the desiredplant. Specific oligonucleotide probes based upon the nucleic acidsequences of the present invention may be designed and synthesized.Moreover, the entire sequences may be used directly to synthesize DNAprobes by methods such as random primer DNA labelling, nick translation,or end-labelling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers may be designed andused to amplify a part or all of the sequences of the present invention.The resulting amplification products may be labelled directly duringamplification reactions or labelled after amplification reactions, andused as probes to isolate full-length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, short segments of the nucleic acids or nucleic acidfragments of the present invention may be used in protocols to amplifylonger nucleic acids or nucleic acid fragments encoding homologous genesfrom DNA or RNA. For example, polymerase chain reaction may be performedon a library of cloned nucleic acid fragments wherein the sequence ofone primer is derived from the nucleic acid sequences of the presentinvention, and the sequence of the other primer takes advantage of thepresence of the polyadenylic acid tracts to the 3′ end of the mRNAprecursor encoding plant genes. Alternatively, the second primersequence may be based upon sequences derived from the cloning vector.For example, those skilled in the art can follow the RACE protocol(Frohman et al. (1988) Proc. Natl. Acad Sci. USA 85:8998, the entiredisclosure of which is incorporated herein by reference) to generatecDNAs by using PCR to amplify copies of the region between a singlepoint in the transcript and the 3′ or 5′ end. Using commerciallyavailable 3′ RACE and 5′ RACE systems (BRL), specific 3′ or 5′ cDNAfragments may be isolated (Ohara et al. (1989) Proc. Natl. Acad Sci USA86:5673; Loh et al. (1989) Science 243:217, the entire disclosures ofwhich are incorporated herein by reference). Products generated by the3′ and 5′ RACE procedures may be combined to generate full-length cDNAs.

In a second aspect of the present invention there is provided asubstantially purified or isolated polypeptide from a clover(Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca)species, selected from the group consisting of C4H and C4H-like, 4CL and4CL-like, LDOX and LDOX-like, TT12 and TT12-like, TT2 and TT2-like, TT8and TT8-like and TTG1 and TTG1-like proteins; and functionally activefragments and variants thereof.

The clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue(Festuca) species may be of any suitable type, including white clover(Trifolium repens), red clover (Trifolium pratense), subterranean clover(Trifolium subterraneum), alfalfa (Medicago sativa), Italian or annualryegrass (Lolium multiflorum), perennial ryegrass (Lolium perenne), tallfescue (Festuca arundinacea), meadow fescue (Festuca pratensis) and redfescue (Festuca rubra). In particular, the species may be a clover or aryegrass, more particularly white clover (T. repens) or perennialryegrass (L. perenne).

In a preferred embodiment of this aspect of the invention, thesubstantially purified or isolated TT12 or TT12-like polypeptideincludes an amino acid sequence selected from the group consisting ofthe sequences shown in FIGS. 2 and 34 hereto, and functionally activefragments and variants thereof.

In a further preferred embodiment of this aspect of the invention, thesubstantially purified or isolated TTG1 or TTG1-like polypeptideincludes an amino acid sequence selected from the group consisting ofthe sequences shown in FIGS. 5 and 38 hereto, and functionally activefragments and variants thereof.

In a further preferred embodiment of this aspect of the invention, thesubstantially purified or isolated TT2 or TT2-like polypeptide includesan amino acid sequence selected from the group consisting of thesequences shown in FIGS. 7, 10, 42 and 45 hereto, and functionallyactive fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention,the substantially purified or isolated TT8 or TT8-like polypeptideincludes an amino acid sequence selected from the group consisting ofthe sequences shown in FIGS. 12 and 49 hereto, and functionally activefragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention,the substantially purified or isolated LDOX or LDOX-like polypeptideincludes an amino acid sequence selected from the group consisting ofthe sequences shown in FIGS. 14 and 53 hereto, and functionally activefragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention,the substantially purified or isolated 4CL or 4CL-like polypeptideincludes an amino acid sequence selected from the group consisting ofthe sequences shown in FIGS. 17, 20, 22, 24, 57, 60, 63 and 66 hereto,and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention,the substantially purified or isolated C4H or C4H-like polypeptideincludes an amino acid sequence selected from the group consisting ofthe sequences shown in FIGS. 26, 29, 31, 71, 75 and 78 hereto, andfunctionally active fragments and variants thereof.

By “functionally active” in relation to polypeptides it is meant thatthe fragment or variant has one or more of the biological properties ofthe proteins TT12, TT12-like, TTG1, TTG1-like, TT2, TT2-like, TT8,TT8-like, LDOX, LDOX-like, 4CL, 4CL-like, C4H, C4H-like, respectively.Additions, deletions, substitutions and derivatizations of one or moreof the amino acids are contemplated so long as the modifications do notresult in loss of functional activity of the fragment or variant.Preferably the functionally active fragment or variant has at leastapproximately 60% identity to the relevant part of the above mentionedamino acid sequence, more preferably at least approximately 80%identity, most preferably at least approximately 90% identity. Suchfunctionally active variants and fragments include, for example, thosehaving conservative amino acid substitutions of one or more residues inthe corresponding amino acid sequence. Preferably the fragment has asize of at least 10 amino acids, more preferably at least 15 aminoacids, most preferably at least 20 amino acids.

In a further embodiment of this aspect of the invention, there isprovided a polypeptide recombinantly produced from a nucleic acid ornucleic acid fragment according to the present invention.

Availability of the nucleotide sequences of the present invention anddeduced amino acid sequences facilitates immunological screening of cDNAexpression libraries. Synthetic peptides representing portions of theinstant amino acid sequences may be synthesized. These peptides may beused to immunise animals to produce polyclonal or monoclonal antibodieswith specificity for peptides and/or proteins including the amino acidsequences. These antibodies may be then used to screen cDNA expressionlibraries to isolate full-length cDNA clones of interest.

A genotype is the genetic constitution of an individual or group.Variations in genotype are important in commercial breeding programs, indetermining parentage, in diagnostics and fingerprinting, and the like.Genotypes can be readily described in terms of genetic markers. Agenetic marker identifies a specific region or locus in the genome. Themore genetic markers, the finer defined is the genotype. A geneticmarker becomes particularly useful when it is allelic between organismsbecause it then may serve to unambiguously identify an individual.Furthermore, a genetic marker becomes particularly useful when it isbased on nucleic acid sequence information that can unambiguouslyestablish a genotype of an individual and when the function encoded bysuch nucleic acid is known and is associated with a specific trait. Suchnucleic acids and/or nucleotide sequence information including singlenucleotide polymorphisms (SNPs), variations in single nucleotidesbetween allelic forms of such nucleotide sequence, may be used asperfect markers or candidate genes for the given trait.

Applicants have identified a number of SNPs of the nucleic acids ornucleic acid fragments of the present invention. These are indicated(marked with grey on the black background) in the figures that showmultiple alignments of nucleotide sequences of nucleic acid fragmentscontributing to consensus contig sequences. See for example, FIGS. 3,15, 18 and 27 hereto.

Accordingly, in a further aspect of the present invention, there isprovided a substantially purified or isolated nucleic acid or nucleicacid fragment including a single nucleotide polymorphism (SNP) from anucleic acid or nucleic acid fragment according to the presentinvention, for example a SNP from a nucleic acid sequence shown in FIGS.3, 15, 18 and 27 hereto; or complements or sequences antisense thereto,and functionally active fragments and variants thereof.

In a still further aspect of the present invention there is provided amethod of isolating a nucleic acid or nucleic acid fragment of thepresent invention including a SNP, said method including sequencingnucleic acid fragments from a nucleic acid library.

The nucleic acid library may be of any suitable type and is preferably acDNA library.

The nucleic acid or nucleic acid fragment may be isolated from arecombinant plasmid or may be amplified, for example using polymerasechain reaction.

The sequencing may be performed by techniques known to those skilled inthe art.

In a still further aspect of the present invention, there is provideduse of the nucleic acids or nucleic acid fragments of the presentinvention including SNPs, and/or nucleotide sequence informationthereof, as molecular genetic markers.

In a still further aspect of the present invention there is provided useof a nucleic acid or nucleic acid fragment of the present invention,and/or nucleotide sequence information thereof, as a molecular geneticmarker.

More particularly, nucleic acids or nucleic acid fragments according tothe present invention and/or nucleotide sequence information thereof maybe used as a molecular genetic marker for quantitative trait loci (QTL)tagging, QTL mapping, DNA fingerprinting and in marker assistedselection, particularly in clovers, alfalfa, ryegrasses and fescues.

Even more particularly, nucleic acids or nucleic acid fragmentsaccording to the present invention and/or nucleotide sequenceinformation thereof may be used as molecular genetic markers in plantimprovement in relation to plant tolerance to biotic stresses such asviruses, microorganisms, insects, fungal pathogens; in relation toforage quality; in relation to bloat safety; in relation to condensedtannin content; in relation to plant pigmentation. Even moreparticularly, sequence information revealing SNPs in allelic variants ofthe nucleic acids or nucleic acid fragments of the present inventionand/or nucleotide sequence information thereof may be used as moleculargenetic markers for QTL tagging and mapping and in marker assistedselection, particularly in clovers, alfalfa, ryegrasses and fescues.

In a still further aspect of the present invention there is provided aconstruct including a nucleic acid or nucleic acid fragment according tothe present invention.

The term “construct” as used herein refers to an artificially assembledor isolated nucleic acid molecule, which includes the gene of interest.In general a construct may include the gene or genes of interest, amarker gene which in some cases can also be the gene of interest andappropriate regulatory sequences. It should be appreciated that theinclusion of regulatory sequences in a construct is optional, forexample, such sequences may not be required in situations where theregulatory sequences of a host cell are to be used. The term constructincludes vectors but should not be seen as being limited thereto.

In a still further aspect of the present invention there is provided avector including a nucleic acid or nucleic acid fragment according tothe present invention.

The term “vector” as used herein encompasses both cloning and expressionvectors. Vectors are often recombinant molecules containing nucleic acidmolecules from several sources.

In a preferred embodiment of this aspect of the invention, the vectormay include a regulatory element such as a promoter, a nucleic acid ornucleic acid fragment according to the present invention and aterminator; said regulatory element, nucleic acid or nucleic acidfragment and terminator being operatively linked.

By “operatively linked” is meant that said regulatory element is capableof causing expression of said nucleic acid or nucleic acid fragment in aplant cell and said terminator is capable of terminating expression ofsaid nucleic acid or nucleic acid fragment in a plant cell. Preferably,said regulatory element is upstream of said nucleic acid or nucleic acidfragment and said terminator is downstream of said nucleic acid ornucleic acid fragment.

The vector may be of any suitable type and may be viral or non-viral.The vector may be an expression vector. Such vectors includechromosomal, non-chromosomal and synthetic nucleic acid sequences, eg.derivatives of plant viruses; bacterial plasmids; derivatives of the Tiplasmid from Agrobacterium tumefaciens, derivatives of the Ri plasmidfrom Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes;bacterial artificial chromosomes; binary bacterial artificialchromosomes; vectors derived from combinations of plasmids and phageDNA. However, any other vector may be used as long as it is replicable,integrative or viable in the plant cell.

The regulatory element and terminator may be of any suitable type andmay be endogenous to the target plant cell or may be exogenous, providedthat they are functional in the target plant cell.

Preferably the regulatory element is a promoter. A variety of promoterswhich may be employed in the vectors of the present invention are wellknown to those skilled in the art. Factors influencing the choice ofpromoter include the desired tissue specificity of the vector, andwhether constitutive or inducible expression is desired and the natureof the plant cell to be transformed (eg. monocotyledon or dicotyledon).Particularly suitable constitutive promoters include the CauliflowerMosaic Virus 35S (CaMV 35S) promoter, the maize Ubiquitin promoter, andthe rice Actin promoter.

A variety of terminators which may be employed in the vectors of thepresent invention are also well known to those skilled in the art. Theterminator may be from the same gene as the promoter sequence or adifferent gene. Particularly suitable terminators are polyadenylationsignals, such as the CaMV 35S polyA and other terminators from thenopaline synthase (nos) and the octopine synthase (ocs) genes.

The vector, in addition to the regulatory element, the nucleic acid ornucleic acid fragment of the present invention and the terminator, mayinclude further elements necessary for expression of the nucleic acid ornucleic acid fragment, in different combinations, for example vectorbackbone, origin of replication (ori), multiple cloning sites, spacersequences, enhancers, introns (such as the maize Ubiquitin Ubi intron),antibiotic resistance genes and other selectable marker genes [such asthe neomycin phosphotransferase (npt2) gene, the hygromycinphosphotransferase (hph) gene, the phosphinothricin acetyltransferase(bar or pat) gene], and reporter genes (such as beta-glucuronidase (GUS)gene (gusA)]. The vector may also contain a ribosome binding site fortranslation initiation. The vector may also include appropriatesequences for amplifying expression.

As an alternative to use of a selectable marker gene to provide aphenotypic trait for selection of transformed host cells, the presenceof the vector in transformed cells may be determined by other techniqueswell known in the art, such as PCR (polymerase chain reaction), Southernblot hybridisation analysis, histochemical GUS assays, northern andwestern blot hybridisation analyses.

Those skilled in the art will appreciate that the various components ofthe vector are operatively linked, so as to result in expression of saidnucleic acid or nucleic acid fragment. Techniques for operativelylinking the components of the vector of the present invention are wellknown to those skilled in the art. Such techniques include the use oflinkers, such as synthetic linkers, for example including one or morerestriction enzyme sites.

The vectors of the present invention may be incorporated into a varietyof plants, including monocotyledons (such as grasses from the generaLolium, Festuca, Paspalum, Pennisetum, Panicum and other forage andturfgrasses, corn, oat, sugarcane, wheat and barley), dicotyledons (suchas arabidopsis, tobacco, clovers, medics, eucalyptus, potato, sugarbeet,canola, soybean, chickpea) and gymnosperms.

In a preferred embodiment, the vectors may be used to transformmonocotyledons, preferably grass species such as ryegrasses (Loliumspecies) and fescues (Festuca species), more preferably perennialryegrass, including forage- and turf-type cultivars. In an alternatepreferred embodiment, the vectors may be used to transform dicotyledons,preferably forage legume species such as clovers (Trifolium species) andmedics (Medicago species), more preferably white clover (Trifoliumrepens), red clover (Trifolium pratense), subterranean clover (Trifoliumsubterraneum) and alfalfa (Medicago sativa). Clovers, alfalfa and medicsare key pasture legumes in temperate climates throughout the world.

Techniques for incorporating the vectors of the present invention intoplant cells (for example by transduction, transfection ortransformation) are well known to those skilled in the art. Suchtechniques include Agrobacterium mediated introduction, electroporationto tissues, cells and protoplasts, protoplast fusion, injection intoreproductive organs, injection into immature embryos and high velocityprojectile introduction to cells, tissues, calli, immature and matureembryos. The choice of technique will depend largely on the type ofplant to be transformed.

Cells incorporating the vectors of the present invention may beselected, as described above, and then cultured in an appropriate mediumto regenerate transformed plants, using techniques well known in theart. The culture conditions, such as temperature, pH and the like, willbe apparent to the person skilled in the art. The resulting plants maybe reproduced, either sexually or asexually, using methods well known inthe art, to produce successive generations of transformed plants.

In a further aspect of the present invention there is provided a plantcell, plant, plant seed or other plant part, including, e.g. transformedwith, a vector or construct, nucleic acid or nucleic acid fragment ofthe present invention.

The plant cell, plant, plant seed or other plant part may be from anysuitable species, including monocotyledons, dicotyledons andgymnosperms. In a preferred embodiment the plant cell, plant, plant seedor other plant part may be from a monocotyledon, preferably a grassspecies, more preferably a ryegrass (Lolium species) or fescue (Festucaspecies), more preferably perennial ryegrass, including both forage- andturf-type cultivars. In an alternate preferred embodiment the plantcell, plant, plant seed or other plant part may be from a dicotyledon,preferably forage legume species such as clovers (Trifolium species) andmedics (Medicago species), more preferably white clover (Trifoliumrepens), red clover (Trifolium pratense), subterranean clover (Trifoliumsubterraneum) and alfalfa (Medicago sativa).

The present invention also provides a plant, plant seed or other plantpart, or a plant extract derived from a plant cell of the presentinvention.

The present invention also provides a plant, plant seed or other plantpart, or a plant extract derived from a plant of the present invention.

Using the methods and materials of the present invention, flavonoidbiosynthesis may be increased or decreased. It may be increased, forexample by incorporating additional copies of a sense nucleic acid ofthe present invention. It may be decreased, for example, byincorporating an antisense nucleic acid or dsRNA or small interferingRNA (siRNA) derived from the nucleotide sequences of the presentinvention. In addition, the number of copies of genes encoding differentenzymes involved in flavonoid biosynthesis may be manipulated to modifyflavonoid biosynthesis, protein binding, metal chelation, antioxidation, UV light absorption, plant pigment production, plant defenseto biotic stresses and modifying forage quality.

In a further aspect of the present invention there is provided a methodof modifying flavonoid biosynthesis; of modifying protein binding, metalchelation, anti-oxidation, and UV-light absorption; of modifying plantpigment production; of modifying plant defense to biotic stresses suchas viruses, microorganisms, insects, fungal pathogens; of modifyingforage quality by disrupting protein foam and conferring protection fromrumen pasture bloat, said method including introducing into said plantan effective amount of a nucleic acid or nucleic acid fragment and/or avector according to the present invention.

By “an effective amount” it is meant an amount sufficient to result inan identifiable phenotypic trait in said plant, or a plant, plant seedor other plant part derived therefrom. Such amounts can be readilydetermined by an appropriately skilled person, taking into account thetype of plant, the route of administration and other relevant factors.Such a person will readily be able to determine a suitable amount andmethod of administration. See, for example, Maniatis et al, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, the entire disclosure of which is incorporated herein byreference.

Using the methods and materials of the present invention, flavonoidbiosynthesis, protein binding, metal chelation, anti-oxidation, UV-lightabsorption, tolerance to biotic stresses such as viruses,microorganisms, insects and fungal pathogens; pigmentation in forexample flowers and leaves; herbage quality and bloat-safety;isoflavonoid content leading to health benefits, may be increased orotherwise altered, for example by incorporating additional copies of asense nucleic acid or nucleic acid fragment of the present invention.They may be decreased or otherwise altered, for example by incorporatingan antisense nucleic acid or nucleic acid fragment of the presentinvention.

Documents cited in this specification are for reference purposes onlyand their inclusion is not acknowledgment that they form part of thecommon general knowledge in the relevant art.

The present invention will now be more fully described with reference tothe accompanying Examples and drawings. It should be understood,however, that the description following is illustrative only and shouldnot be taken in any way as a restriction on the generality of theinvention described above.

IN THE FIGURES

FIG. 1 shows the consensus nucleotide sequence of WcCTa (TrTT12a) (SEQID No: 1).

FIG. 2 shows the deduced amino acid sequence of WcCTa (TrTT12a) (SEQ IDNo: 2).

FIG. 3 shows the nucleotide sequences of nucleic acid fragmentscontributing to the consensus sequence of WcCTa (TrTT12a) (SEQ ID Nos: 3to 6).

FIG. 4 shows the nucleotide sequence of WcCTb (TrTTG1a) (SEQ ID No: 7).

FIG. 5 shows the deduced amino acid sequence of WcCTb (TrTTG1a) (SEQ IDNo: 8).

FIG. 6 shows the consensus nucleotide sequence of WcCTc (TrTT2a) (SEQ IDNo: 9).

FIG. 7 shows the deduced amino acid sequence of WcCTc (TrTT2a) (SEQ IDNo: 10).

FIG. 8 shows the nucleotide sequences of nucleic acid fragmentscontributing to the consensus sequence of WcCTc (TrTT2b) (SEQ ID Nos: 11and 12).

FIG. 9 shows the nucleotide sequence of WcCTd (TrTT2b) (SEQ ID No: 13).

FIG. 10 shows the deduced amino acid sequence of WcCTd (TrTT2b) (SEQ IDNo: 14).

FIG. 11 shows the nucleotide sequence of WcCTe (TrTT8a) (SEQ ID No: 15).

FIG. 12 shows the deduced amino acid sequence of WcCTe (TrTT8a) (SEQ IDNo: 16).

FIG. 13 shows the consensus nucleotide sequence of WcCTf (TrLDOXa) (SEQID No: 17).

FIG. 14 shows the deduced amino acid sequence of WcCTf (TrLDOXa) (SEQ IDNo: 18).

FIG. 15 shows the nucleotide sequences of nucleic acid fragmentscontributing to the consensus sequence of WcCTf (TrLDOXa) (SEQ ID Nos:19 to 33).

FIG. 16 shows the consensus nucleotide sequence of WcCTg (Tr4CLa) (SEQID No: 34).

FIG. 17 shows the deduced amino acid sequence of WcCTg (Tr4CLa) (SEQ IDNo: 35).

FIG. 18 shows the nucleotide sequences of nucleic acid fragmentscontributing to the consensus sequence of WcCTg (Tr4CLa) (SEQ ID Nos: 36to 38).

FIG. 19 shows the nucleotide sequence of WcCTh (Tr4CLb) (SEQ ID No: 39).

FIG. 20 shows the deduced amino acid sequence of WcCTh (Tr4CLb) (SEQ IDNo: 40).

FIG. 21 shows the nucleotide sequence of WcCTi (Tr4CLc) (SEQ ID No: 41).

FIG. 22 shows the deduced amino acid sequence of WcCTi (Tr4CLc) (SEQ IDNo: 42).

FIG. 23 shows the nucleotide sequence of WcCTj (Tr4CLd) (SEQ ID No: 43).

FIG. 24 shows the deduced amino acid sequence of WcCTj (Tr4CLd) (SEQ IDNo: 44).

FIG. 25 shows the consensus nucleotide sequence of WcCTk (TrC4Ha) (SEQID No: 45).

FIG. 26 shows the deduced amino acid sequence of WcCTk (TrC4Ha) (SEQ IDNo: 46).

FIG. 27 shows the nucleotide sequences of nucleic acid fragmentscontributing to the consensus sequence of WcCTk (TrC4Ha) (SEQ ID Nos: 47to 51).

FIG. 28 shows the nucleotide sequence of WCCTl (TrC4Hb) (SEQ ID No: 52).

FIG. 29 shows the deduced amino acid sequence of WcCTl (TrC4Hb) (SEQ IDNo: 53).

FIG. 30 shows the nucleotide sequence of WcCTm (TrC4Hc) (SEQ ID No: 54).

FIG. 31 shows the deduced amino acid sequence of WcCTm (TrC4Hc) (SEQ IDNo: 55).

FIG. 32 shows a plasmid map of the cDNA encoding white clover WcCTa(TrTT12a).

FIG. 33 shows the full nucleotide sequence of the white clover WcCTa(TrTT12a) cDNA (SEQ ID No: 56).

FIG. 34 shows the deduced amino acid sequence of white clover WcCTa(TrTT12a) cDNA (SEQ ID No: 57).

FIG. 35 shows plasmid maps of the cDNA encoding white clover WcCTa(TrTT12a) in the sense and antisense orientations in the pPZP221 binarytransformation vector

FIG. 36 shows a plasmid map of the cDNA encoding white clover WcCTb(TrTTG1a).

FIG. 37 shows the full nucleotide sequence of the white clover WcCTb(TrTTG1a) cDNA (SEQ ID No: 58).

FIG. 38 shows the deduced amino acid sequence of the white clover WcCTb(TrTTG1a) cDNA (SEQ ID No: 59).

FIG. 39 shows plasmid maps of the cDNA encoding white clover WcCTb(TrTTG1a) in the sense and antisense orientations in the pPZP221 binarytransformation vector

FIG. 40 shows a plasmid map of the cDNA encoding white clover WcCTc(TrTT2a).

FIG. 41 shows the full nucleotide sequence of the white clover WcCTc(TrTT2a) CDNA (SEQ ID No: 60).

FIG. 42 shows the deduced amino acid sequence of the white clover WcCTc(TrTT2a) cDNA (SEQ ID No: 61).

FIG. 43 shows a plasmid map of the cDNA encoding white clover WcCTd(TrTT2b).

FIG. 44 shows the full nucleotide sequence of the white clover WcCTd(TrTT2b) cDNA (SEQ ID No: 62).

FIG. 45 shows the deduced amino acid sequence of the white clover WcCTd(TrTT2b) cDNA (SEQ ID No: 63).

FIG. 46 shows plasmid maps of the cDNAs encoding white clover WcCTc(TrTT2a) and WcCTd (TrTT2b) in the sense and antisense orientations inthe pPZP221 binary transformation vector

FIG. 47 shows a plasmid map of the cDNA encoding white clover WcCTe(TrTT8a).

FIG. 48 shows the full nucleotide sequence of the white clover WcCTe(TrTT8a) cDNA (SEQ ID No: 64).

FIG. 49 shows the deduced amino acid sequence of the white clover WcCTe(TrTT8a) cDNA (SEQ ID No: 65).

FIG. 50 shows a plasmid map of the cDNA encoding white clover WcCTe(TrTT8a) in the antisense orientation in the pPZP221 binarytransformation vector

FIG. 51 shows a plasmid map of the cDNA encoding white clover WcCTf(TrLDOXa).

FIG. 52 shows the full nucleotide sequence of the white clover WcCTf(TrLDOXa) cDNA (SEQ ID No: 66).

FIG. 53 shows the deduced amino acid sequence of the white clover WcCTf(TrLDOXa) cDNA (SEQ ID No: 67).

FIG. 54 shows plasmid maps of the cDNA encoding white clover WcCTf(TrLDOXa) in the sense and antisense orientations in the pPZP221 binarytransformation vector

FIG. 55 shows a plasmid map of the cDNA encoding white clover WcCTg(Tr4CLa).

FIG. 56 shows the full nucleotide sequence of the white clover WcCTg(Tr4CLa) cDNA (SEQ ID No: 68).

FIG. 57 shows the deduced amino acid sequence of the white clover WcCTg(Tr4CLa) cDNA (SEQ ID No: 69).

FIG. 58 shows a plasmid map of the cDNA encoding white clover WcCTh(Tr4CLb).

FIG. 59 shows the full nucleotide sequence of the white clover WcCTh(Tr4CLb) cDNA (SEQ ID No: 70).

FIG. 60 shows the deduced amino acid sequence of the white clover WcCTh(Tr4CLb) cDNA (SEQ ID No: 71).

FIG. 61 shows a plasmid map of the cDNA encoding white clover WcCTi(Tr4CLc).

FIG. 62 shows the full nucleotide sequence of the white clover WcCTi(Tr4CLc) cDNA (SEQ ID No: 72).

FIG. 63 shows the deduced amino acid sequence of the white clover WcCTi(Tr4CLc) cDNA (SEQ ID No: 73).

FIG. 64 shows a plasmid map of the cDNA encoding white clover WcCTj(Tr4CLd).

FIG. 65 shows the full nucleotide sequence of the white clover WcCTj(Tr4CLd) cDNA (SEQ ID No: 74).

FIG. 66 shows the deduced amino acid sequence of the white clover WcCTj(Tr4CLd) cDNA (SEQ ID No: 75).

FIG. 67 shows plasmid maps of the cDNAs encoding white clover WcCTg(Tr4CLa), WcCTh (Tr4CLb), WcCTi (Tr4CLc) and WcCTj (Tr4CLd) in the senseorientation in the pPZP221 binary transformation vector

FIG. 68 shows plasmid maps of the cDNAs encoding white WcCTg (Tr4CLa),WcCTh (Tr4CLb), WcCTi (Tr4CLc) and WcCTj (Tr4CLd) in the antisenseorientation in the pPZP221 binary transformation vector

FIG. 69 shows a plasmid map of the cDNA encoding white clover WcCTk(TrC4Ha).

FIG. 70 shows the full nucleotide sequence of the white clover WcCTk(TrC4Ha) cDNA (SEQ ID No: 76).

FIG. 71 shows the deduced amino acid sequence of the white clover WcCTk(TrC4Ha) cDNA (SEQ ID No: 77).

FIG. 72 shows a plasmid map of the cDNA encoding white clover WcCTk(TrC4Ha) in the sense orientation in the pPZP221 binary transformationvector

FIG. 73 shows a plasmid map of the cDNA encoding white clover WcCTl(TrC4Hb).

FIG. 74 shows the full nucleotide sequence of the white clover WcCTl(TrC4Hb) cDNA (SEQ ID No: 78).

FIG. 75 shows the deduced amino acid sequence of the white clover WcCTl(TrC4Hb) cDNA (SEQ ID No: 79).

FIG. 76 shows a plasmid map of the cDNA encoding white clover WcCTm(TrC4Hc).

FIG. 77 shows the full nucleotide sequence of the white clover WcCTm(TrC4Hc) cDNA (SEQ ID No: 80).

FIG. 78 shows the deduced amino acid sequence of the white clover WcCTm(TrC4Hc) cDNA (SEQ ID No. 81)

FIG. 79 shows plasmid maps of the cDNAs encoding white clover WcCTk(TrC4Ha), WCCTI (TrC4Hb) and WcCTm (TrC4Hc) in the antisense orientationin the pPZP221 binary transformation vector

FIG. 80 shows a plasmid map of the pDONR221 GATEWAY entry vector(Invitrogen, Carlsbad, USA).

FIG. 81 shows the steps of selection during Agrobacterium-mediatedtransformation of white clover cotyledons. Cotyledonary explants areextracted from imbibed seeds (A), cocultivated with Agrobacteriumtumefaciens strain containing the binary transformation vector andsubjected to a series of 2-week selective steps on tissue culture plates(B, C and D). Shoots are excised and grown on root-inducing media intissue culture vessels (E). Finally, transgenic white clover plantletsare transferred to glasshouse conditions (F and G), allowing molecularand phenotypic analyses to take place.

FIG. 82 shows 4-dimethylaminocinnemaldehyde (DMACA) staining patterns inTrifolium repens (cv ‘Mink’) leaf (A) and inflorescence (B) tissue andin Lotus corniculatus (cv ‘Draco’) leaf tissue (C).

FIG. 83 shows the results of real-time RT-PCR analysis of white cloverhomologues of TT12, TTG1, TT2, TT8, LDOX, 4CL and C4H in upper and lowerhalves of white clover (cv Mink) buds as well as whole buds. Moreparticularly, FIG. 77 shows comparative expression of flavonoid-relatedgenes relative to a histone control gene. Complementary DNA from whiteclover (cv Mink) upper, lower and whole buds was tested by real-timeRT-PCT using SYBR Green chemistry, primer sets designed using cDNAclones of flavonoid-related genes (Table 4) and the δδCT method ofanalysis. TT12, TTG1, TT2b, TT8, LDOX, 4Cla, 4CLb, 4CLd, C4Ha, C4Hb andC4Hc correspond to WcCTa, WcCTb, WcCTd, WcCTe, WcCTf, WcCTg, WcCTh,WcCTj, WcCTK, WcCT1, and WcCTM respectively.

EXAMPLE 1

Preparation of cDNA Libraries, Isolation and Sequencing of cDNAs Codingfor TT12-like, TTG1-like, TT2-like, TT8-like, LDOX, LDOX-like, 4CL,4CL-like, C4H and C4H-like Proteins from White Clover (Trifolium Repens)

cDNA libraries representing mRNAs from various organs and tissues ofwhite clover (Trifolium repens) were prepared. The characteristics ofthe white clover libraries, respectively, are described below (Tables 1and 2).

TABLE 1 cDNA libraries from white clover (Trifolium repens) LibraryOrgan/Tissue 01wc Whole seedling, light grown 02wc Nodulated root 3, 5,10, 14, 21 &28 day old seedling 03wc Nodules pinched off roots of 42 dayold rhizobium inoculated plants 04wc Cut leaf and stem collected after0, 1, 4, 6 &14 h after cutting 05wc Inflorescences: <50% open, not fullyopen and fully open 06wc Dark grown etiolated 07wc Inflorescence - veryearly stages, stem elongation, <15 petals, 15-20 petals 08wc seed frozenat −80° C., imbibed in dark overnight at 10° C. 09wc Drought stressedplants 10wc AMV infected leaf 11wc WCMV infected leaf 12wc Phophorusstarved plants 13wc Vegetative stolon tip 14wc stolon root initials 15wcSenescing stolon 16wc Senescing leaf

The cDNA libraries may be prepared by any of many methods available. Forexample, total RNA may be isolated using the Trizol method (Gibco-BRL,USA) or the RNeasy Plant Mini kit (Qiagen, Germany), following themanufacturers' instructions. cDNAs may be generated using the SMART PCRcDNA synthesis kit (Clontech, USA), cDNAs may be amplified by longdistance polymerase chain reaction using the Advantage 2 PCR Enzymesystem (Clontech, USA), cDNAs may be cleaned using the GeneClean spincolumn (Bio 101, USA), tailed and size fractionated, according to theprotocol provided by Clontech. The cDNAs may be introduced into thepGEM-T Easy Vector system 1 (Promega, USA) according to the protocolprovided by Promega. The cDNAs in the pGEM-T Easy plasmid vector aretransfected into Escherichia coli Epicurian coli XL10-Gold ultracompetent cells (Stratagene, USA) according to the protocol provided byStratagene.

Alternatively, the cDNAs may be introduced into plasmid vectors forfirst preparing the cDNA libraries in Uni-ZAP XR vectors according tothe manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif., USA). The Uni-ZAP XR libraries are converted into plasmidlibraries according to the protocol provided by Stratagene. Uponconversion, cDNA inserts will be contained in the plasmid vectorpBluescript. In addition, the cDNAs may be introduced directly intoprecut pBluescript II SK(+) vectors (Stratagene) using T4 DNA ligase(New England Biolabs), followed by transfection into E. coli DH10B cellsaccording to the manufacturer's protocol (GIBCO BRL Products).

Once the cDNA inserts are in plasmid vectors, plasmid DNAs are preparedfrom randomly picked bacterial colonies containing recombinant plasmids,or the insert cDNA sequences are amplified via polymerase chain reactionusing primers specific for vector sequences flanking the inserted cDNAsequences. Plasmid DNA preparation may be performed robotically usingthe Qiagen QiaPrep Turbo kit (Qiagen, Germany) according to the protocolprovided by Qiagen. Amplified insert DNAs are sequenced indye-terminator sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”). The resulting ESTs are analyzedusing an Applied Biosystems ABI 3700 sequence analyser.

EXAMPLE 2 DNA Sequence Analyses

The cDNA clones encoding TT12, TT12-like, TTG1, TTG1-like, TT8,TT8-like, TT2, TT2-like, LDOX, LDOX-like, 4CL, 4CL-like, C4H andC4H-like proteins were identified by conducting BLAST (Basic LocalAlignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410)searches. The cDNA sequences obtained were analysed for similarity toall publicly available DNA sequences contained in the eBioinformaticsnucleotide database using the BLASTN algorithm provided by the NationalCenter for Biotechnology Information (NCBI). The DNA sequences weretranslated in all reading frames and compared for similarity to allpublicly available protein sequences contained in the SWISS-PROT proteinsequence database using BLASTX algorithm (v 2.0.1) (Gish and States(1993) Nature Genetics 3:266-272) provided by the NCBI.

The cDNA sequences obtained and identified were then used to identifyadditional identical and/or overlapping cDNA sequences generated usingthe BLASTN algorithm. The identical and/or overlapping sequences weresubjected to a multiple alignment using the CLUSTALw algorithm, and togenerate a consensus contig sequence derived from this multiple sequencealignment. The consensus contig sequence was then used as a query for asearch against the SWISS-PROT protein sequence database using the BLASTXalgorithm to confirm the initial identification.

EXAMPLE 3

Identification and Full-Length Sequencing of cDNAs Encoding White CloverTT12, TTG1, TT2, TT8, LDOX, 4CL and C4H Proteins

To fully characterise for the purposes of the generation of probes forhybridisation experiments and the generation of transformation vectors,a set of cDNAs encoding white clover TT12, TTG1, TT2, TT8, LDOX, 4CL andC4H proteins was identified and fully sequenced.

Full-length or partial cDNAs were identified from our EST sequencedatabase using relevant published sequences (NCBI databank) as queriesfor BLAST searches. Full-length cDNAs were identified by alignment ofthe query and hit sequences using Sequencher (Gene Codes Corp., AnnArbor, Mich. 48108, USA). The original cDNA in the pGEM-T easy vectorwas then used to transform chemically competent DH5 alpha cells(Invitrogen, Carlsbad, USA). At least two colonies per transformationwere picked for initial sequencing with M13F and M13R primers. Theresulting sequences were aligned with the original EST sequence usingSequencher to confirm identity and one of the two clones was picked forfull-length sequencing, usually the one with the best initial sequencingresult.

Sequencing was completed by primer walking, i.e. oligonucleotide primerswere designed to the initial sequence and used for further sequencingfrom the 5′ end. The sequences of the oligonucleotide primers are shownin Table 2. In most instances, an extended poly-A tail necessitated thesequencing of the cDNA to be completed from the 5′ end.

Contigs were then assembled in Sequencher. The contigs include at leastthe 5′ end of the original EST sequence and extend to at least thepoly-A tail at the 3′ end of the cDNA.

Plasmid maps and the full or partial cDNA sequences of white cloverTT12, TTG1, TT2, TT8, LDOX, 4CL and C4H genes in the pGEM-T Easy vectorwere obtained (FIGS. 32, 33, 36, 37, 40, 41, 43, 44, 47, 48, 51, 52, 55,56, 58, 59, 61, 62, 64, 65, 69, 70, 73, 74, 76, 77).

TABLE 2 List of primers used for sequencing of the full-length cDNAssequencing primer sequence gene name clone ID primer (5′>3′) WcCTa05wc1CsD12 05wc1CsD12.f GCATTTGCATTGAGTTGTC (TrTT12a) 05wc1CsD12.f2AGCCAGTGTGCGAGTTAG 05wc1CsD12.f3 AATTGTCAGTCTTCGTAGTG 05wc1CsD12.r1ACAACGAAGTATGACAGAAG WcCTb 10wc1CsD07 10wc1CsD07.f GCATCGCTGTTGGTAGTT(TrTTG1a) 10wc1CsD07.r1 CAACGCCTCTTTCAATGTC 10wc1CsD07.f2TACCCCTTTGCTTCGTTTG WcCTc 14wc1LsB05 14wc1LsB05.f1 CACACGCATTTGAAGAAG(TrTT2a) WcCTd 04wc1EsE11 04wc1EsE11.f1 AACCAACAAGGCCACAAC (TrTT2b)WcCTe 06wc2DsD04 06wc2DsD04.f1 ATAGGTGAGACAAGGAGACAGA (TrTT8a) WcCTf07wc3GsD03 07wc3GsD03.f1 GCCTAAGACTCCAGCTGA (TrLDOXa) 07wc3GsD03.r1TCCCATTCAAGTTGACCAC 07wc3GsD03.f2 AACAAGGGCCACAAGTTC 07wc3GsD03.f3TCTTGGGCAGTGTTTTGTG WcCTg 14wc2KsH10 14wc2KsH10.f1 CAGCAGCCAATCCTTTCTTC(Tr4Cla) 14wc2KsH10.f2 AGTCCAACAGGGTGATGT 14wc2KsH10.f3GTAGTTCCTCCGATAGTGT 14wc2ksH10.f4 TCTGATGCTGCTGTTGTC WcCTh 13wc1DsH0713wc1DsH07.f1 TTGGTAAGGAACTTGAGGACA (Tr4CLb) 13wc1DsH07.f2CAAAAGCCTCCAATGCTAAG WcCTi 16wc1NsB11 16wc1NsB11.f1 GAAGAGGCTGTAAAGGAG(Tr4CLc) WcCTj 12wc1CsA11 12wc1CsA11.f1 ACTCATCGTAACTCAATCC (Tr4CLd)12wc1CsA11.f2 GCGTTGGTAAAAAGTGGTG 12wc1CsA11.f3 TTTCGATGCTGCTGTTGT12wc1CsA11.f4 GCCTATTCGTTCGCTTCT WcCTk 14wc2CsB09 14wc2CsB09.f1TACGGTGAACATTGGCGT (TrC4Ha) 14wc2CsB09.f2 GATGCTCAAAAGAAAGGAGAG14wc2CsB09.f3 ATCGGGCGTCTTGTTCAG WcCTl 11wc1OsE04 11wc1OsE04.f1AGGACCAGGACACCAAGTA (TrC4Hb) WcCTm 06wc1OsE12 06wc1OsE12.f1TAACCCGGCTCTATGGAA (TrC4Hc) (810)

EXAMPLE 4

Development of Binary Transformation Vectors Containing Chimeric Geneswith cDNA Sequences from White Clover TT12a, TrTTG1, TrTT2a, TrTT2b,TrTT8a, TrLDOXa, Tr4CLa, Tr4CLb, Tr4Clc Tr4CLd, TrC4Ha, TrC4Hb andTrC4Hc

To alter the expression of the proteins involved in flavonoidbiosynthesis, protein binding, metal chelation, anti-oxidation, UV-lightabsorption, tolerance to biotic stresses such as viruses,micro-organisms, insects and fungal pathogens; pigmentation in forexample flowers and leaves; herbage quality and bloat-safety andisoflavonoid content leading to health benefits, white clover TT12a,TTG1, TT2a, TT2b, TT8a, LDOXa, 4CLa, 4CLb, 4Clc 4CLd, C4Ha, C4Hb andC4Hc through antisense and/or sense suppression technology and forover-expression of these key proteins in transgenic plants, a set ofsense and antisense binary transformation vectors was produced.

cDNA fragments were generated by high fidelity PCR using the originalpGEM-T Easy plasmid cDNA as a template. The primers used (Table 3)contained attB1 and attB2 GATEWAY® recombination sites for directionalcloning into the target vector. After PCR amplification and purificationof the products, the CDNA fragments were cloned into the recombinationsite of the pDONR221™ vector (FIG. 80) using BP GATEWAY® technology(Invitrogen, Carlsbad, USA). vector The pPZP221 binary vector(Hajdukiewicz et al., 1994) was modified to contain the 35S2 cassettefrom pKYLX71:35S² as follows. pKYLX71:35S² was cut with Clal. The 5′overhang was filled in using Klenow and the blunt end was A-tailed withTaq polymerase. After cutting with EcoRI, the 2kb fragment with anEcoRI-compatible and a 3′-A tail was gel-purified. pPZP221 was cut withHindIII and the resulting 5′ overhang filled in and T-tailed with Taqpolymerase. The remainder of the original pPZP221 multi-cloning site wasremoved by digestion with EcoRI, and the expression cassette cloned intothe EcoRI site and the 3′ T overhang restoring the HindIII site. Thisbinary vector contains between the left and right border the plantselectable marker gene aaaC1 under the control of the 35S promoter and35S terminator and the pKYLX71:35S²-derived expression cassette with aCaMV 35S promoter with a duplicated enhancer region and an rbcSterminator. This vector was GATEWAY®-enabled by digesting it with Xbaland blunt-ended using Klenow DNA polymerase, allowing the RfArecombination cassette to be cloned in the sense or antisenseorientation between the enhanced 35S promoter and the rbcS terminator.

The orientation of the constructs (sense or antisense) was checked byrestriction enzyme digestion and sequencing. Transformation vectorscontaining chimeric genes using full-length open reading frame cDNAsencoding white clover TT12a, TTG1, TT2a, TT2b, TT8a, LDOXa, 4CLa,Tr4CLb, 4Clc 4CLd, C4Ha, C4Hb and C4Hc proteins in sense and antisenseorientations under the control of the CaMV 35S² promoter were generated(FIGS. 35, 39, 46, 50, 54, 67, 68, 72 and 79).

TABLE 3 List of primers used to PCR-amplify the open reading frames offlavonoid-related genes from white clover gene name clone ID primerprimer sequence (5′->3′) WcCTa 05wc1CsD12 05wc1CsD12GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (TrTT12a) CATGAGCTCTATAGAAAACCAACC WcCTa05wc1CsD12 05wc1CsD12GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (TrTT12a)TCATATGTCGGCAACCAGTTGATCC WcCTb 10wc1CsD07 10wc1CsD07GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (TrTTG1a) CATGGAGAATTCAACTCAAGAATCACACWcCTb 10wc1CsD07 10wc1CsD07GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (TrTT2a)TCAAACCCGCAAAAGCTGCATCTTG WcCTc 14wc1LsB05 14wc1LsB05GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (TrTT2a) CATGGTAAGAGCTCCTTGTTGTGA WcCTc14wc1LsB05 14wc1LsB05GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (TrTT2a)TTAGAACTCTGGCAATTCTATTTGATC WcCTd 04wc1EsE11 04wc1EsE11GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (TrTT2b) CATGGTGAGAGCTCCATGTTGTGA WcCTd04wc1EsE11 04wc1EsE11GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (TrTT2b)TCACAATTCAAGTAACTCAGTAATTTCC WcCTe* 06wc2DsD04 06wc2DsD04GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (TrTT8a) CATGAACCATGTTTTGTCAGAAAGAAGGWcCTe* 06wc2DsD04 06wc2DsD04GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (TrTT8a)TCAAAACTTTGAAGCCACTTTTTGTAGG WcCTf 07wc3GsD03 07wc3GsD03GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (TrLDOXa) CATGGGAGCCGTGGCACAAAGAGTTGWcCTf 07wc3GsD03 07wc3GsD03GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (TrLDOXa)TCATTTTTTAGGATCATCCTTCTTCTC WcCTg 14wc2KsH10 14wc2KsH10GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (Tr4CLa) CATGGCGGCCGCGGGAATTCGATTAAGCWcCTg 14wc2ksH10 14wc2KsH10GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (Tr4CLa)TTATTCTGCTGCTAACTTTGCTCTGAG WcCTh 13wc1DsH07 13wc1DsH07GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (Tr4CLb) CATGGCGGCCGCGGGAATTCGATTAAGCWcCTh 13wc1DsH07 13wc1DsH07GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (Tr4CLb)TTAATTTGTTGGAACACCAGCTGC WcCTi 16wc1NsB11 16wc1NsB11GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (Tr4CLc) CATGGCGGCCGCGGGAATTCGATTAAGCWcCTi 16wc1NsB11 16wc1NsB11GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (Tr4CLc)TCAAGGCTTTTGGGTGGTACTTTCTAAC WcCTj 12wc1CsA11 12wc1CsA11GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (Tr4CLd) CATGTCACCATTTCCTCCACAGCAAG WcCTj12wc1CsA11 12wc1CsA11GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (Tr4CLd)TTAAGTGGCCACCACCAAACCTTCG WcCTk 14wc2CsB09 14wc2CsB09GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (TrC4Ha) CATGGATCTACTCCTTCTTGAAAAGACTCWcCTk 14wc2CsB09 14wc2CsB09GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (TrC4Ha)TTAAAATGATCTTGGCTTAGCAACAATG WcCTl* 11wc1OsE04 11wo1OsE04GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (TrC4Hb) CGCAGTGGTAACAACGCAGAGTACGCWcCTl* 11wc1OsE04 11wc1OsE04GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (TrC4Hb)TTAAAATGATCTTGGCTTAGCAACAATG WcCTm* 06wc1OsE12 06wc1OsE12GW.fGGGGACAAGTTTGTACAAAAAAGCAGGCTT (TrC4Hc) CCCGACGTCGCATGCTCCCGGC WcCTm*06wc1OsE12 06wc1OsE12GW.r GGGGACCACTTTGTACAAGAAAGCTGGGTC (TrC4Hc)TTAAAATGATCTTGGCTTAGCAACAATG

EXAMPLE 5 Production and Analysis of Transgenic White Clover PlantsCarrying Chimeric White Clover TT12a, TTG1, TT2a, TT2b, TT8a, LDOXa,4CLa, 4CLb, 4Clc 4CLd, C4Ha, C4Hb and C4Hc Genes Involved in FlavonoidBiosynthesis

A set of transgenic white clover plants carrying white clover genesinvolved in flavonoid biosynthesis, protein binding, metal chelation,anti-oxidation, UV-light absorption, tolerance to biotic stresses suchas viruses, micro-organisms, insects and fungal pathogens; pigmentationin for example flowers and leaves; herbage quality and bloat-safety andisoflavonoid content leading to health benefits, were produced.

pPZP221-based transformation vectors with WcCTa (TrTT12a), WcCTb(TrTTG1), WcCTc (TrTT2a), WcCTd (TrTT2b), WcCTe (TrTT8a), WcCTf(TrLDOXa), WcCTg (Tr4Cla), WcCTh (Tr4CLb), WcCTi (Tr4Clc) WcCTj(Tr4CLd), WcCTk (TrC4Ha), WcCTl (TrC4Hb) and WcCTm (TrC4Hc) cDNAscomprising the full open reading frame sequences in sense and antisenseorientations under the control of the CaMV 35S promoter with duplicatedenhancer region (35S²) were generated as detailed in Example 4.

Agrobacterium-mediated gene transfer experiments were performed usingthese transformation vectors.

The production of transgenic white clover plants carrying the whiteclover WcCTa (TrTT12a), WcCTb (TrTTG1), WcCTc (TrTT2a), WcCTd (TrTT2b),WcCTe (TrTT8a), WcCTf (TrLDOXa), WcCTg (Tr4Cla), WcCTh (Tr4CLb), WcCTi(Tr4Clc), WcCTj (Tr4CLd), WcCTk (TrC4Ha), WcCTl (TrC4Hb) and WcCTm(TrC4Hc) cDNAs under the control of the CaMV 35S promoter withduplicated enhancer region (35S²) is described here in detail. Theselection process is shown in FIG. 81.

Preparation of White Clover Cotyledonary Explants

White clover (cv ‘Mink’) seeds were rinsed for 5 minutes in running tapwater and incubated twice, for 5 minutes in 70% v/v ethanol in a 120 mltissue culture container with gentle shaking. The same container wasused to incubate the seeds for 2 minutes in 1% sodium hypochlorite (1:3ratio of Domestos™ bleach in water) with gentle shaking. The seeds werethen rinsed six times in sterile water in a laminar flow hood andincubated for 18 hours at 4° C. in the dark. Cotyledonary explant wereextracted using 10 ml syringes attached to 21 G needles (Terumo, Japan)under a dissecting microscope in a laminar flow hood. Both layers of theseed coat were peeled away, the end of the hypocotyl was cut off and thecotyledons with approximately 4 mm of hypocotyl were separated andtransferred to a 90×90×20 mm petri dish containing MGL medium.

Preparation of Agrobacterium

Agrobacterium tumefaciens strain AGL-1 containing each PZP221-derivedbinary expression vector was streaked on LB medium containing 50 μg/mlrifampicin and 100 pg/ml spectinomycin and grown at 27° C. for 48 hours.A single colony was used to inoculate 5 ml of LB medium containing 50μg/ml rifampicin and 100 μg/ml spectinomycin and grown over night at 27°C. and 250 rpm on an orbital shaker. The overnight culture was used asan inoculum for 40 ml of YEP medium containing 100 μg/ml spectinomycinand 40 mg/l acetosyringone. Incubation was over night at 27° C. and 250rpm on an orbital shaker in a 250 ml Erlenmeyer flask.

The overnight cultures were centrifuged for 15 min at 5500×g and thesupernatant discarded. The cells were resuspended in MGL media with 40mg/l acetosyringone to a volume corresponding to an OD₆₀₀ reading of0.4. The cells were then incubated at 27° C. and 250 rpm until the OD₆₀₀reading reached 0.8.

Cocultivation and Selection of White Clover Transformants

The MGL medium was removed from the petri dish containing white clovercotyledonary explants and replaced with the prepared Agrobacteriumsuspension using a sterile serological pipette. The petri dish wassealed with laboratory film, covered with aluminium foil and incubatedwith gentle shaking for 45 min. The dish was opened in the laminar flowhood and the Agrobacterium suspension removed with a pipette. Theexplants were then transferred to plates containing RM73 media with 40mg/l acetosyringone (Table 4) and incubated for 3 days in a plant tissueculture room at 22° C. with a 16 hour photoperiod. After this, theexplants were transferred, with the hypocotyl end in the media, toplates containing RM73 media with 75 mg/l gentamicin and 250 mg/lcefotaxime. The explants were transferred to fresh plates every twoweeks for 6-8 weeks. Shoots were then transferred to 120 ml tissueculture vessels containing RIM media (Table 5) with 75 mg/l gentamicinand 250 mg/l cefotaxime. When roots had developed, the plantlets weretransferred to pots of soil and after 2 weeks of recovery in a mistingbench, were grown under standard glasshouse conditions.

Preparation of Genomic DNA

1-2 leaflets of white clover plants recovered from the transformationprocess were harvested and freeze-dried. The tissue was homogenised on aRetsch MM300 mixer mill, then centrifuged for 10 min at 1700×g tocollect cell debris. Genomic DNA was isolated from the supernatant usingWizard Magnetic 96 DNA Plant System kits (Promega) on a Biomek FX(Beckman Coulter). 5 μl of the sample (50 μl) were then analysed on anagarose gel to check the yield and the quality of the genomic DNA.

Analysis of DNA From Putative Transgenic Lines Using Real-Time PCR

Genomic DNA was analysed for the presence of the transgene by real-timePCR using SYBR Green chemistry. PCR primer pairs were designed to detectthe aacC1 gentamycin resistance gene in the transferred T-DNA regionusing MacVector (Accelrys). The sequences of these primers are asfollows:

pPZPaacC1-1.f 5′-TCAAGTATGGGCATCATTCGCAC-3′ pPZPaacC1-1.r5′-TGCTCAAACCGGGCAGAACG-3′

2.5 μl of each genomic DNA sample was run in a 25 μl PCR reactionincluding SYBR Green on an ABI (Applied Biosystems) together withsamples containing DNA isolated from wild type white clover plants (cv‘Mink’, negative control), samples containing buffer instead of DNA(buffer control) and samples containing the plasmid used fortransformation (positive plasmid control).

TABLE 4 Composition of RM73 tissue culture media, pH 5.75 Component[Stock] For 1 litre MS Macronutients  10× 100 mL MS Micronutrients 100×10 mL MS Vitamins 100× 10 mL TDZ 100 mM 50 uL NAA  1 mM 0.5 mL Sucrose(BDH Chemicals) — 30 g Agar — 8 g

TABLE 5 Composition of root-inducing tissue culture media (RIM73), pH5.75 Component [Stock] For 1 litre MS macronutrients  10× 100 mL MSmicronutrients 100× 10 mL MS vitamins 100× 10 mL Indole-3-butyric acid 1mM 1.2 mL Sucrose (BDH Chemicals) — 15 g Agar (Becton-Dickinson) — 8 g

EXAMPLE 6

Analysis of Condensed Tannins and Their Monomers in the Leaves ofTransgenic White Clover Plants Carrying Chimeric White Clover TT12a,TTG1, TT2a, TT2b, TT8a, LDOXa, 4CLa, 4CLb, 4Clc 4CLd, C4Ha, C4Hb andC4Hc genes involved in Flavonoid Biosynthesis

Accumulation of condensed tannins and their monomers was analysedqualitatively in leaves of transgenic and wild type (cv ‘Mink’) whiteclover plants using 4-dimethylaminocinnemaldehyde (DMACA) staining. Twomature leaflets from each plant were decolourised in absolute ethanol in6-well tissue culture plates for 3 hours with gentle shaking. Theethanol was removed and replaced with a 0.01% w/v solution of DMACA(Fluka), freshly made up in absolute ethanol with 2.4% v/v concentratedhydrochloric acid. After 1 hour of incubation with gentle shaking, theleaflets were rinsed with distilled water and mounted in 50% glycerolfor analysis with a dissecting microscope. Wild type white clover plantsshow blue staining in epidermal cells in the floral organs and intrichomes. Lotus comiculatus (cv ‘Draco’), a forage legume with a‘bloat-safe’ level of condensed tannins in the leaves, shows bluestaining of approximately 50% of mesophyll cells in leaves (FIG. 82).Achieving a level of condensed tannins in white clover leaves that iscomparable to the level seen in leaves of L. comiculatus by metabolicengineering would be agronomically valuable.

DMACA staining can detect economically significant levels of condensedtannins and their monomers in the leaves of established bloat-safeforage legumes. However, the condensation of catechin monomers to formcondensed tannins and their transport from the cytoplasm to the vacuoleis poorly understood. Hence, modifying the regulation of known enzymesand transcription factors in the flavonoid pathway may up-regulatecatechin levels but not increase condensed tannin levels, and therefore,bloat-safety. The PVPP-butanol-HCl assay detects only condensed tannins,relying on the ability of condensed tannins, but not their monomers tobind to PVPP. The detailed method is as follows.

Clover leaf and inflorescence (positive control) tissue was snap-frozenand ground to a fine powder in a mortar and pestle under liquidnitrogen. After grinding, 0.75 g of the powder from each sample wastransferred to a 14 ml screw-cap centrifuge tube (Falcon), vortex-mixedwith 1.5 ml of extraction buffer containing 80% v/v methanol indistilled water with 5.3 mM sodium bisulfite. Samples were mixed for 5hours on a mixing wheel before centrifugation at 3000×g for 10 minutes.A 1 ml aliquot of each supernatant was transferred to a 1.5 mlmicrocentrifuge tube and reduced to 0.25 ml in a vacuum centrifuge.Equal volumes of the sample were added to each of two 1.5 mlmicrocentrifuge tubes containing 25 mg of polyvinyl polypyrrolidone(PVPP). Each mixture was vortex-mixed intermittently for 15 min andcentrifuged for 1 min at maximum speed in a microcentrifuge. Afterremoval of the supernatant, the pellet was washed four times with 1 mlof methanol, with a 1 min centrifugation step at maximum speed in amicrocentrifuge between each wash. A freshly-made 70:30 (v/v) solutionof butanol and concentrated hydrochloric acid was added to each pelletand one tube of the mixture was incubated for 1 hour at 70° C., whereasthe other tube was incubated at ambient temperature. The difference inthe absorbance (530 nm) between the two tubes from each plant sample wasproportional to the level of condensed tannins in the sample. This assaycan be quantitated with a condensed tannin of known concentration,although only the relative levels of tannins were measured in thisexperiment.

EXAMPLE 7 Design of Real Time RT-PCR Primers Based on cDNA Sequences ofClover TT12, TTG1, TT2, TT8, LDOX, 4CL and C4H Genes

Real-time RT-PCR is a recently developed technique that allows morequantitative analyses of gene expression than Northern or conventionalRT-PCR experiments. Essentially, real-time RT-PCR with SYBR Greenchemistry and gene-specific primers involves the automatic measurementof the level of a fluorescent PCR product generated from a cDNA speciesover each cycle. The abundance of each template is proportional to theamplification rate. Therefore, a threshold corresponding to the start ofthe exponential phase of PCR allows the relative abundance of targetgenes to be standardised against a uniformly expressed ‘housekeeping’gene in each tissue and compared to a negative control without atemplate. Real-time RT-PCR with SYBR Green chemistry has been usedsuccessfully by others in the field to quantify the expression of fourflavonoid-related genes in Lotus corniculatus plants exposed todifferent light regimes (Paolocci et al., 2005)

A Real-Time RT-PCR strategy involving with SYBR Green chemistry and theδδCT method of analysis was used characterise the expression of TT12,TTG1, TT2, TT8, LDOX, 4CL and C4H homologues in white clover tissuescontaining high and low levels of condensed tannins. This approach aimedto determine which of the genes and isoforms were most likely to beinvolved in condensed tannin production, or in the production of otherflavonoids, and could therefore be targeted for overexpression ordownregulation in the metabolic engineering of bloat-safe white clover.

The full-length CDNA sequences of white clover of TT12, TTG1, TT2, TT8,LDOX, 4CL and C4H homologues were used as input data for the PrimerExpress (Applied Biosystems, Foster City, USA) primer design program,using the default settings, no 3′ GC clamp and a predicted amplicon sizeof 50-150 base pairs. Primers close to the 3′ ends of the inputsequences were preferred, due to the likelihood of a large number ofcDNA molecules derived from clover samples being incomplete at the 5′end. The sequences of the chosen primers are shown in Table 6.

The specificity of the primer sets was tested using 1 ul of plasmid DNA(0.01 ng/ul) from the original cDNA cloned into pGEM-T Easy orautoclaved, purified water, 12.5 ul 2× SYBR Green Master Mix (AppliedBiosystems), 0.5 ul each of the forward and reverse primers (10 uM) and10.5 ul of autoclaved, purified water (Sartorius AG, Goettingen,Germany). Real-time PCR was performed in 96-well optical PCR plates(Applied Biosystems) using the Stratagene MX3000P cycler and thefollowing cycling parameters: 95° C. for 10 min, 40 cycles of 95° C. for30 sec and 60° C. for 1 min, followed by 55° C. for 1 min and 95° C. for1 min. All of the primer sets except those designed to amplify cloverTT2a amplified a satisfactory level of products from the correspondingcDNA templates with a cycle threshold cut-off of 24 cycles (Table 7).The primer sets were isoform-specific, with the exception of the twosets designed to amplify clover C4H homologues.

It was shown by DMACA staining that the lower half of Mink white cloverbuds are enriched for condensed tannins. Therefore a preliminaryexperiment was carried out to test for the expression of clover TT12,TTG1, TT2, TT8, LDOX, 4CL and C4H genes in the buds of white clover (cvMink), relative to expression of a clover histone control gene. TotalRNA was extracted from upper and lower halves of buds as well as wholebuds using the RNeasy kit (QIAGEN GmbH, Hilden, Germany) andcontaminating genomic DNA was digested on the column using the optionalon-column DNAse digestion according to the manufacturers' instructions.Complementary DNA (cDNA) was synthesised from 0.5 ug of total RNA usingthe Quantitect Reverse Transcriptase Kit (QIAGEN GmbH). Real-time RT-PCRreactions were set up and run as described earlier using 1 ul of cDNA,plasmid control DNA or autoclaved, purified water as the template. Theexperiment showed that expression of clover LDOX correlated well withcondensed tannin production in the lower half of white clover buds (FIG.83).

TABLE 6 List of primers designed for Real-time RT-PCR analysis ofcondensed tannin-rich organs of white clover, based on cDNA sequences ofclover TT12, TTG1, TT2, TT8, LDOX, 4CL and C4H genes Gene name Clone IDprimer 1 (forward) primer 2 (reverse) WcCTa 05wc1CsD12GACAGAGAGCATAGCCGAGCA GGTATAAGACCGCGAGCGAA (TrTT12a) WcCTb 10wc1CsD07AACTCATGTTCCATCCCGCA CGGAGGAGGTTTTCTGGAGAG (TrTTG1a) WcCTc 14wc1LsB05GTAATGGCAACTGGCGTGCT CACATCTTAACAAGCCTCGTAGCT (TrTT2a) WcCTd 04wc1EsE11CCATTCTAATTGGCGTGCTCTT CCACACCTTAACAACCCAGCTT (TrTT2b) WcCTe 06wc2DsD04TGGGAGGCTTCATGTGATCA GCATTAGCTGGTCCTTTGAACTTAG (TrTT8a) WcCTf 07wc3GsD03GCTAGTGGTCAACTTGAATGGGA TCAGGAAAAATACAATGAAAGAAATAATCT (TrLDOXa) WcCTg14wc2KsH10 GCACCCACCGGAAAAGTCTA CCGAGAGGTGAGTTCGACGT (Tr4Cla) WcCTh13wc1DsH07 TCATAGTGGATAGGCTTAAAGAATTGAT TGGGATGTGAAAGAATAATGGCTT(Tr4CLb) WcCTi 16wc1NsB11 GTTGTCCCGCAAAAGGATGT CACAAAGGCAACAGGAACTTCAC(Tr4CLc) WcCTj 12wc1CsA11 CTTTCCTCGGTGCCTCCTTC AAGGATTTGCGGTGGTGATG(Tr4CLd) WcCTk 14wc2CsB09 CTTGCCGGTTATGACATCCC CCACGCGTTGACCAATATCTT(TrC4Ha) WcCTm 06wc1OsE12 (TrC4Hc) WcCTl 11wc1OsE04CGTTGATGAGAGAAAGAAACTTGAAA GAGCATCCAAAATGTGATCAATTG (TrC4Hb)

TABLE 7 Results of testing real-time PCR primer sets on plasmidscontaining cDNA sequences encoding clover TT12, TTG1, TT2, TT8, LDOX,4CL and C4H genes Primers Template TT12a TTG1a TT2a TT2b TT8a LDOXa 4CLa4CLb 4CLc 4CLd C4Hac C4Hb WcCTa 26.7 (TrTT12) WcCTb 19.6 (TrTTG1a) WcCTc27.7 0 Ct (TrTT2a) WcCTd 36.2 20.8 (TrTT2b) WcCTe 20 (TrTT8) WcCTf 21.13(TrLDOX) WcCTg 19.5 no Ct 37.7 no Ct (Tr4CLa) WcCTh no Ct 19.3 39.7 noCt (Tr4CLb) WcCTi 37.4 36.8 19.8 35.8 (Tr4CLc) WcCTj 31.3 31.8 32.5 20.6(Tr4CLd) WcCTk 22.44 22.9  (TrC4ha) WoCTI 22.05 17.55 (TrC4Hb) WcCTm20.2 37.13 (TrC4Hc) ddH2O 37.2 0 Ct 0 Ct 38.8 35.3 0 Ct 37.6 0 Ct 32.531.1 37.2 0 Ct

REFERENCES

-   Causier, B. and Davies B. (2002). Analysing protein-protein    interactions with the yeast two-hybrid system. Plant Mol. Biol. 50:    855-870-   Frohman et al. (1988) Rapid production of full-length cDNAs from    rare transcripts: amplification using a single gene-specific    oligonucleotide primer. Proc. Natl. Acad Sci. USA 85:8998-   Gish and States (1993) Identification of protein coding regions by    database similarity search. Nature Genetics 3:266-272-   Hink, M A, Bisseling, T. and Visser, A. G. (2002). Imaging    protein-protein interactions in living cells. Plant Mol. Biol.    50:871-873-   Loh, E. Y., Elliott, J. F., Cwirla, S., Lanier, L. L., Davis, M. M.    (1989). Polymerase chain reaction with single-sided specificity:    Analysis of T-cell receptor delta chain. Science 243:217-220-   Ohara, O., Dorit, R. L., Gilbert, W. (1989). One-sided polymerase    chain reaction: The amplification of cDNA. Proc. Natl. Acad Sci USA    86:5673-5677-   Paolocci, F., Bovone, T. Tosti, N., Arcioni, S. and Damiani, F.    (2005). Light and an exogenous transcription factor qualitatively    and quantitatively affect the biosynthetic pathway of condensed    tannins in Lotus corniculatus leaves. J. Exp. Bot. 56: 1093-1103

Finally, it is to be understood that various alterations, modificationsand/or additions may be made without departing from the spirit of thepresent invention as outlined herein.

It will also be understood that the term “comprises” (or its grammaticalvariants) as used in this specification is equivalent to the term“includes” and should not be taken as excluding the presence of otherelements or features.

Documents cited in this specification are for reference purposes onlyand their inclusion is not acknowledgment that they form part of thecommon general knowledge in the relevant art.

1. A substantially purified or isolated nucleic acid or nucleic acidfragment encoding a protein selected from the group consisting ofTRANSPARENT TESTA GLABRA 1 (TTG1), TRANSPARENT TESTA 2 (TT2),TRANSPARENT TESTA 8 (TT8), leucoanthocyanidin dioxygenase (LDOX),cinnamate-4-hydroxylase (C4H), 4-coumaroyl:CoA-ligase (4CL), andTRANSPARENT TESTA 12 (TT12); from a clover (Trifolium), medic(Medicago), ryegrass (Lolium) or fescue (Festuca) species; or afunctionally active fragment or variant thereof.
 2. A nucleic acid ornucleic acid fragment according to claim 1 wherein said protein is froma clover species.
 3. A nucleic acid or nucleic acid fragment accordingto claim 2, wherein said clover species is white clover (Trifoliumrepens).
 4. A nucleic acid or nucleic acid fragment according to claim1, encoding a TT12 or TT12-like protein, or complementary or antisenseto a sequence encoding a TT12 or TT12-like protein, and comprising anucleotide sequence selected from the group consisting of (a) SequenceID Nos: 1, 3 to 6 and 56, (b) complements of the sequences recited in(a); (c) sequences antisense to the sequences recited in (a) and (b);and (d) functionally active fragments and variants of the sequencesrecited in (a), (b) and (c).
 5. A nucleic acid or nucleic acid fragmentaccording to claim 1 encoding a TTG 1 or TTG 1-like protein, orcomplementary or antisense to a sequence encoding a TTG1 or TTG1-likeprotein, and comprising a nucleotide sequence selected from the groupconsisting of (a) Sequence ID Nos: 7 and 58; (b) complements of thesequences recited in (a); (c) sequences antisense to the sequencesrecited in (a) and (b); and (d) functionally active fragments andvariants of the sequences recited in (a), (b) and (c).
 6. A nucleic acidor nucleic acid fragment according to claim 1 , encoding a TT2 orTT2-like protein, or complementary or antisense to a sequence encoding aTT2 or TT2-like protein, and comprising a nucleotide sequence selectedfrom the group consisting of (a) Sequence ID Nos: 9, 11 and 12, 13, 60and 62; (b) complements of the sequences recited in (a); (c) sequencesantisense to the sequences recited in (a) and (b); and (d) functionallyactive fragments and variants of the sequences recited in (a), (b) and(c).
 7. A nucleic acid or nucleic acid fragment according to claim 1encoding a TT8 or TT8-like protein, or complementary or antisense to asequence encoding a TT8 or TT8-like protein, and comprising a nucleotidesequence selected from the group consisting of (a) Sequence ID Nos: 15and 64; (b) complements of the sequences recited in (a); (c) sequencesantisense to the sequences recited in (a) and (b); and (d) functionallyactive fragments and variants of the sequences recited in (a), (b) and(c).
 8. A nucleic acid or nucleic acid fragment according to claim 1 ,encoding an LDOX or LDOX-like protein, or complementary or antisense toa sequence encoding a LDOX or LDOX-like protein, and comprising anucleotide sequence selected from the group consisting of (a) SequenceID Nos: 17, 19 to 33 and 66; (b) complements of the sequences recited in(a); (c) sequences antisense to the sequences recited in (a) and (b);and (d) functionally active fragments and variants of the sequencesrecited in (a), (b) and (c).
 9. A nucleic acid or nucleic acid fragmentaccording to claim 1 encoding a 4CL or 4CL-like protein, orcomplementary or antisense to a sequence encoding a 4CL or 4CL-likeprotein, and comprising a nucleotide sequence selected from the groupconsisting of (a) Sequence ID Nos: 34, 36 to 38, 39, 41, 43, 68, 70, 72and 74; (b) complements of the sequences recited in (a); (c) sequencesantisense to the sequences recited in (a) and (b); and (d) functionallyactive fragments and variants of the sequences recited in (a), (b) and(c).
 10. A nucleic acid or nucleic acid fragment according to claim 1,encoding a C4H or C4H-like protein, or complementary or antisense to asequence encoding a C4H or C4H-like protein, and comprising a nucleotidesequence selected from the group consisting of (a) Sequence ID Nos: 45,47 to 51, 52, 54, 76, 78 and 80; (b) complements of the sequencesrecited in (a); (c) sequences antisense to the sequences recited in (a)and (b); and (d) functionally active fragments and variants of thesequences recited in (a), (b) and (c).
 11. A construct comprising anucleic acid or nucleic acid fragment according to claim
 1. 12. A vectorcomprising a nucleic acid or nucleic acid fragment according to claim 1.13. A vector according to claim 12, further including a promoter and aterminator, said promoter, nucleic acid or nucleic acid fragment andterminator being operatively linked.
 14. A plant cell, plant, plant seedor other plant part, comprising a nucleotide construct or a vector, saidconstruct or vector comprising a nucleic acid or nucleic acid fragmentaccording to claim
 1. 15. A plant, plant seed or other plant partderived from a plant cell or plant according to claim
 14. 16. A methodaccording to claim 34 wherein the property of the plant that is modifiedis flavonoid biosynthesis in a plant, said method comprising introducinginto said plant an effective flavonoid biosynthesis-modifying amount ofthe nucleic acid or nucleic acid fragment.
 17. A method according toclaim 34 wherein the property of the plant that is modified is proteinbinding, metal chelation, anti-oxidation, and/or UV-light absorption ina plant, said method comprising introducing into said plant an effectiveprotein binding, metal chelation, anti-oxidation, and/or UV-lightabsorption modifying amount of the nucleic acid or nucleic acidfragment.
 18. A method according to claim 34 wherein the property of theplant that is modified is pigment production in a plant, said methodcomprising introducing into said plant an effective pigmentproduction-modifying amount of the nucleic acid or nucleic acidfragment.
 19. A method according to claim 34 wherein the property of theplant that is modified is plant defense to a biotic stress, said methodcomprising introducing into said plant an effective defense to bioticstress-modifying amount of the nucleic acid or nucleic acid fragment.20. A method according to claim 19 wherein said biotic stress isselected from the group consisting of viruses, microorganisms, insectsand fungal pathogens.
 21. A method according to claim 34 wherein theproperty of the plant that is modified is forage quality of a plant bydisrupting protein foam and/or conferring protection from rumen pasturebloat, said method comprising introducing into said plant an effectiveforage quality-modifying amount of the nucleic acid or nucleic acidfragment.
 22. Use of a nucleic acid or nucleic acid fragment accordingto claim 1, and/or nucleotide sequence information thereof, and/orsingle nucleotide polymorphisms thereof as a molecular genetic marker.23. A substantially purified or isolated polypeptide from a clover(Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca)species, selected from the group consisting of TT12 and TT12-like, TT2and TT2-like, TT8 and TT8-like, LDOX and LDOX-like, C4H and C4H-like,4CL and 4CL-like and TT12 and TT12-like; and functionally activefragments and variants thereof.
 24. A polypeptide according to claim 23wherein said polypeptide is from a clover species
 25. A polypeptideaccording to claim 24, wherein said clover species is white clover(Trifolium repens).
 26. A polypeptide according to claim 23, whereinsaid polypeptide is TT12 or TT12-like and comprises an amino acidsequence selected from the group consisting of Sequence ID Nos: 2 and57; and functionally active fragments and variants thereof.
 27. Apolypeptide according to claim 23, wherein said polypeptide is TTG1 orTTG1-like and comprises an amino acid sequence selected from the groupconsisting of Sequence ID Nos: 8 and 59; and functionally activefragments and variants thereof.
 28. A polypeptide according to claim 23,wherein said polypeptide is TT2 or TT2-like and comprises an amino acidsequence selected from the group consisting of Sequence ID Nos: 10, 14,61, 63; and functionally active fragments and variants thereof.
 29. Apolypeptide according to claim 23, wherein said polypeptide is TT8 orTT8-like and comprises an amino acid sequence selected from the groupconsisting of Sequence ID Nos: 16 and 65; and functionally activefragments and variants thereof.
 30. A polypeptide according to claim 23,wherein said polypeptide is LDOX or LDOX-like and comprises an aminoacid sequence selected from the group consisting of Sequence ID Nos: 18and 67; and functionally active fragments and variants thereof.
 31. Apolypeptide according to claim 23, wherein said polypeptide is 4CL or4CL-like and comprises an amino acid sequence selected from the groupconsisting of Sequence ID Nos: 35, 40, 42, 44, 69, 71, 73 and 75; andfunctionally active fragments and variants thereof.
 32. A polypeptideaccording to claim 23, wherein said polypeptide is C4H or C4H-like andcomprises an amino acid sequence selected from the group consisting ofSequence ID Nos: 46, 53, 55, 60, 64 and 81; and functionally activefragments and variants thereof.
 33. A substantially purified or isolatedpolypeptide encoded by a nucleic acid or nucleic acid fragment accordingto claim
 1. 34. A method of modifying a flavonoid-related biologicalproperty of a plant comprising introducing into said plant an effectiveamount of a nucleic acid or nucleic acid fragment according to claim 1,wherein said nucleic acid or nucleic acid fragment is optionallyintroduced in a construct or vector.